Preclinical study of experimental burns treated with photobiomodulation and Human Amniotic Membrane, both isolated and associated

Abstract Objective: to evaluate the effect of photobiomodulation with low-level 660 nm laser alone or associated with Human Amniotic Membrane in the repair of partial-thickness burns in rats. Method: an experimental study conducted with 48 male Wistar rats, randomized into four groups: Control, Human Amniotic Membrane, Low-Level Laser Therapy, and Low-Level Laser Therapy associated with Human Amniotic Membrane. The histopathological characteristics of the skin samples were analyzed 7 and 14 days after the burn. The data obtained were submitted to the Kolmogorov-Smirnov and Mann-Whitney tests. Results: the histological analysis of the burn injuries showed a decrease in inflammation (p<0.0001) and an increase in proliferation of fibroblasts (p<0.0001) mainly at 7 days in all treatments related to the control group. At 14 days, the greater effectiveness in accelerating the healing process was significant (p<0.0001) in the Low-Level Laser Therapy group associated with the Human Amniotic Membrane. Conclusion: the association of photobiomodulation therapies with the Human Amniotic Membrane allowed verifying a reduction in the healing process time of the experimental lesions, stimulating its proposal as a treatment protocol in partial-thickness burns.


Introduction
Skin burns are injuries caused by heat, radiation, radioactivity, electricity, friction or contact with chemical products. In the world, nearly 180,000 people die every year as a result of this problem, a reality also expressed in the last decade in Brazil by the high in-hospital mortality rate due to this cause (1)(2) .
Thermal burns can occur by scalds (hot liquids), contact (hot solids) or flames (1) . In addition to being the most prevalent and strenuous, these types of lesions directly impair the phases of an adequate healing process, as they present reduced angiogenesis, sustained inflammation, oxidative stress, increased proteolysis and septicemia as main characteristics (3) .
As for depth, burns can be classified as follows: superficial-thickness (first degree), partial-superficial (second degree), deep-superficial (second degree) and full-thickness (third or fourth degree). Histologically, superficial-thickness burns only reach the epidermis; partial superficial-thickness burns reach the epidermis and papillary dermis, but the skin annexes remain intact; deep superficial-thickness burns injure the epidermis and reticular dermis and most of the skin appendages are destroyed; and, in full-thickness burns, the entire epidermis, dermis and appendages of the skin are destroyed (third degree), and may even involve the muscular fascia and/or bone (fourth degree) (4) .
The main clinical characteristics of partial superficialthickness lesions are erythema, phlictenes, humidity, hyperemia, pressure pallor and healing time from 7 to 20 days (4) . In this sense, the healing process of burn injuries is complex, as it involves differentiated cells that are activated during the different and overlapping phases of the tissue repair process called inflammation, proliferation and remodeling (5) .
The injuries resulting from burns establish challenges in the skin repair process, as the burned area presents characteristics that hinder repair, such as irregular edges and tissue necrosis, in addition to being capable of reaching the epidermis, dermis and deep tissues. The need for hospitalizations and high hospital costs is also highlighted (6) .
The multifaceted environment of burn wound healing has stimulated the investigation of innovative therapeutic interventions that enable immediate repair of this problem (7) . Thus, defining an appropriate strategy in view of the needs and complexity of burns becomes fundamental for the success of therapeutic treatments in terms of performance and cost. In this sense, biomaterials and new technologies stand out for having general properties capable of inducing different biological responses that can be adapted according to the application (8) .
In the context of technologies, photobiomodulation with the use of low-level laser therapy (LLLT) has stood out for favoring wound healing due to its biomodular effects (9)(10)(11) . In contrast, related to biomaterials, the Human Amniotic Membrane (HAM) has been used as a promising alternative, with great potential for application in regenerative medicine for presenting low antigenicity and protection against infections, as well as for acting as a substrate for epithelization (12) . Therefore, several studies have clinically evaluated the benefit of HAM as a biological substitute (13)(14)(15) .
Thus, considering the complexity of burn therapy and the need for experimental studies that investigate alternative treatments that favor tissue regeneration in this condition, this study evaluated the effect of LLLT associated with HAM in the repair of superficial partial thickness burns in rats.

Type of study
This is an experimental research study with a quantitative approach.

Study locus
The research was conducted at the experimental surgery laboratory of the UNINOVAFAPI University Center, located in the municipality of Teresina (PI),

Study period
Data collection took place from January to March 2019.

Animals
A total of 48 male rats (Rattus norvegicus albinus, Wistar) were studied: 40 days old, weighing 200 ± 50 g, kept in polypropylene cages under aseptic conditions, specific feed with food and water ad libitum, and exposed to a 12/12-hour light-dark cycle, housed in individual cages.

Study groups
The animals were randomized and allocated into four groups with twelve animals each, namely: Control

Data collection
The experimental protocol was developed in five stages: capture of the human placenta, processing of the biomaterial, induction of burns, and application of HAM fragments and LLLT, isolated or associated.
The placentas were collected from two selected parturients, after signing the Free and Informed Consent Form (FICF), subjected to elective cesarean section, with a healthy clinical history, negative serological tests for HIV-1, VDRL, HbsAg and anti-HCV, and gestational age from 37 weeks to 41 weeks and 6 days (full term placenta), according to criteria established in a previous study (16) .
The placentas were inspected immediately after removal, placed in a sterile plastic bag, packed at a temperature of 10ºC and transported to the experimental surgery laboratory. The biomaterial was processed in an aseptic environment following the protocols described (16) , isolating the HAM that was sectioned into fragments of suitable dimensions for this research (4 x 4 cm) that were used in 24 hours (17) .
Initially, the animals were weighed, sedated (Xylazine 2%, 0.01 mL/kg and Ketamine 10%, 0.005 mL/kg) and their dorsal region was epilated. The experimental burn was induced using a beaker (3 cm in diameter), filled with 50 mL of water heated to 100°C, supported in direct contact with the shaved region skin for 10 seconds, without additional pressure. Subsequently, the lesions were evaluated considering the macroscopic aspects, which included observation of staining (red or pink) and presence of a bubble to characterize the superficial partial thickness burn (4) .
The animals from group C received no treatment.
In the animals from the HAM and LLLT+HAM groups, HAM fragments were applied immediately after the burn, always with the mesenchymal face in contact with the skin lesion area, exceeding its edges by 1 cm, and fixed with a topical adhesive. In the HAM+LLLT group, the protocols described for the HAM and LLLT groups were associated, and the laser was applied on the amniotic membrane every 24 hours at both experimental times studied (7 and 14 days).
The animals were euthanized according to

Histological techniques
The burned skin area and the surrounding area, collagen type I -yellow-reddish color; and collagen type III -green-whitish color. All histomorphometric analyses were performed blindly.

Statistical analysis
The data collected were evaluated for the

Discussion
Using rats in experimental research involving tissue repair in burns has been a frequent practice, especially because their skin composition (epidermis and dermis) is similar to that of human skin, in addition to presenting low cost and reduced healing times. However, morphology of the rodent skin is unique and differs from the architecture  in treatment (7,(10)(11)19) .
In our research it was verified that, at the We discovered that all therapies (both isolated and associated) were effective in reducing inflammatory cells at both experimental times; such findings reveal the importance of their early use so that the tissue repair process occurs without delays. In this sense, a study with induced acute wounds revealed that exacerbated and prolonged inflammation causes harms in the reepithelialization process by modifying the formation of granulation tissue, with an increase in the possibility of scar formation (20) .
It is worth noting that, in the intergroup analysis of the second experimental period, LLLT+HAM was extremely effective when compared to the isolated therapies. In addition to that, LLLT alone was more effective in reducing the mean number of inflammatory cells than the treatment of burns with HAM alone.
Photobiomodulation with LLLT has been used to reduce inflammation, pain and edema, as well as to preserve and restore tissues damaged by the injury. These effects can be achieved using wavelengths between 600 and 1000 nm (21) . In this sense, clinical and experimental studies with partial and total thickness burns have used photobiomodulation with LLLT with a wavelength of 660 nm, ratifying choice of this parameter also in our study (5,19,(22)(23) .
Similarly to the results found in our study, modulation of the inflammatory response was evidenced in the healing of skin grafts in rats in a recent study that used the same LLLT irradiation protocol (24) . In addition to that and corroborating our findings, the effects with a single LLLT dose have been pointed out in the literature, with acceleration of the inflammatory phase in skin repair among them (22) .

It is known that inflammation and angiogenesis
are important factors in determining wound healing and that the decrease in inflammation enables an increase in angiogenesis. Thus, the modulation properties of the inflammation and cell proliferation levels are found in research studies with LLLT (25)(26) .
In the context of the association of LLLT with other therapies in burn treatments, a study that used this tool combined with medicinal honey obtained results

for inflammation and pain attenuation in burn healing
and acceleration of the repair process characterized by increased cell proliferation (7) , corroborating the same effects of the therapeutic association protocol used in our study.
It is noted that, for the treatment of superficial partial thickness burns, the ideal dressings are those that can preserve heat, provide moisture, avoid contamination by microorganisms, be safe and not adhere to the injury or require frequent exchanges (27) . Therefore, HAM stands out for being a biomaterial that has all the listed characteristics (12) .
HAM has been applied to acute and chronic wounds, as evidenced by the promising results obtained with the application of this biomaterial in the healing of these lesions due to its properties (28)(29) . It is noted that the dressings used with this biomaterial or in association with other products can facilitate proliferation of fibroblasts and contribute to the release of angiogenic factors (30) .
In the intergroup analysis context and regarding proliferation of fibroblasts, our results show that This fact evidences the high capacity to repair the burned tissue when choosing the treatment with the associated therapies.
A number of studies evidence a biological response favoring the tissue repair process by stimulating proliferation of fibroblasts, and the improvement in microcirculation has also been proven in the context of photobiomodulation with LLLT (31)(32)(33) .
The data obtained in our study reinforce the beneficial properties of using LLLT and HAM reported in previous studies in the context of the isolated use of these therapies or their association with other products (7,(30)(31)(32)(33)(34) . However,